Evaluation of possible biological control of Fusarium concentricum sp. using plant extracts and antagonistic species of microbes in vitro [version 2; peer review: 1 approved, 2 approved with reservations]

Background: Fusarium concentricum is one of the most devastating fungi responsible for fruit and vegetable crops rot worldwide. The present study was designed to find an ecofriendly control measure for pathogenic Fusarium concentricum , using suitable bioagents. Methods: Medicinal plant extracts were evaluated or their antifungal activities against Fusarium concentricum using the poisoned food and serial dilution methods. Antagonistic potency of some nonpathogenic microbes was also assessed on Fusarium concentricum using the dual culture method. Results: Highest inhibition of growth of Fusarium concentricum was observed with 68.1% (0.389 mg per 90 mm Petri plate) of mycelia on Coccinia grandis plant leaf extract, in comparison to the control grown with 100.0% (1.22 mg/dish). The tested plants extract showed MIC values rages of 80-150 µg/ml on the isolated fungus. The highest inhibition of radial growth was observed using Trichoderma viride on Fusarium concentricum (46.01% inhibition). Conclusions: The findings of present study would be benevolent for antifungal drug development to control Fusarium concentricum causing fruit and vegetable rot. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. dealt with an important topic in the safe control of the fungus Fusarium sp. that causes damage to many types of fruits without the use of chemicals that represent a danger to the environment and health. Authors presented the antifungal activity of some plant extract as a eco-friendly control measure for pathogenic Fusarium species using the poisoned food method. They found some plant extracts showed promising (>65%) inhibitory activity against Fusarium sp. and suggested to use as The authors are reporting some interesting results which will be of significance to the readership however, the manuscript lacks indepth research. The effect of the bacteria and botanical extracts should be tested in the pot studies for their effectiveness. In vitro studies alone will not warrant their suitability to be used as bioinoculants for management of pathogens. Without the in vivo studies the work appears as shallow research. The authors may be encouraged to do pot studies and update the manuscript.


Introduction
Fusarium species are a large genus of hyaline filamentous mold fungi, responsible for fruit and vegetable crop rot (Al-Najada & Gherbawy, 2015;Ziedan et al., 2018). Fusarium species are deeply invasive and can cause hematogenously disseminated infections with high mortality in neutropenic patients (Dignani & Anaissie, 2004). Numerous species of Fusarium contribute to yield loss and reduced quality to varying degrees by infection with some mycotoxins (O'Donnell et al., 2009). They also cause decay of various fruit in storage and postharvest conditions (Whiteside et al., 1988). The fruit rot caused by Fusarium incurs enormous yield losses and is often observed in fields and markets (Baria et al., 2015). The application of antagonistic agents and plant extracts in agriculture are becoming a major focus of plant protection research. Different preservatives or fungicide treatments are frequently applied to manage fruits diseases and decay, which is an alarming health concern (Munhuweyi et al., 2020). Since most chemical fungicides are highly toxic to humans and animals and they frequently cause water and soil pollution (Al-Najada & Gherbawy, 2015). Moreover, continuous and indiscriminate use is leading to the development of fungicide resistant strains of pathogens (Tafinta et al., 2013). There are some previous reports on plants extract based controlling techniques in Fusarium fungi causing diseases for some crops and plants ( Baria et al., 2015;Ziedan et al., 2018).
In Bangladesh, fruit rot is a destructive disease caused by Fusarium species on pre-harvest and postharvest fruits. To the best of our knowledge, there is no previous research on biological control of this pathogenic fungus. Therefore, the main objective of the study was to find an ecofriendly control system of Fusarium concentricum to decrease fruit and vegetable rot in Bangladesh. Collected plants were washed with water to remove dust from the plants' surface and dried in room temperature. Plant extract preparation and fractionation was performed according to the method by Kader et al. (2018). Different parts of selected plants-bulb of Allium sativum; rhizome of Zingiber officinale; leaves of Coccinia grandis, Brassica juncea, Ocimum tenuiflorum; and flowers of Hibiscus rosa-sinensis plants were cut into small species and ground by blender to form fine powder. The dried powder of the plants (100gm of each plant) were rinsed in methanol (500ml) using a conical flask, and were incubated in a shaking incubator with occasional shaking for fourteen days. The liquid contents were pressed through Markin cloth followed by filtration using Whatman no. 1 filter paper. Obtained filtered liquids were dehydrated in vacuo to leave a blackish and sticky mass. The extracts were collected in vials and preserved in a refrigerator at 4°C.

Collection of Fusarium concentricum
The inhibitory effect of different plant extracts was measured by following the poisoned food technique (Balamurugan, 2014). For this, 20µg of each plant extract was added to 20ml of potato dextrose agar (PDA) to fill a 90mm size Petri plate and mixed well. After solidification, seven day old 6 mm size fungal plug was placed in the center of the Petri plates. The Petri plates were incubated at 35°C for seven days in static condition.

Determination of minimum inhibitory concentration (MIC)
The required MIC values of selected plants extracts were measured through the serial dilution technique (Hussain et al., 2010). The extract of 1 gm of plant-based methanol leaves was dissolved into 1 ml methanol to obtain this same stock solution. Fungal suspension was taken into each test tube and added 50-150µg of plants extracts. The tested tubes were then incubated for 3 days. The progression of the isolated fungus was detected in tubes in which the strength of the substances was just below the inhibition level.

In vitro antagonistic test
For evaluation of antagonistic effects, six non-pathogenic pure microbe cultures, Escherichia coli (pathogenic), Rhizobium phaseoli, Rhizobium leguminosarum, Neofusicoccum mangifera, Trichoderma viride and Pestalotiopsis sp. were used against Fusarium concentricum. The pure microbe cultures were kindly provided by Dr. Md. Salah Uddin, Associate Professor and Director, Microbiology Lab., Department of Genetic Engineering and Biotechnology, University of Rajshahi, as the part of collaboration.

Amendments from Version 1
Changes to the manuscript include:

REVISED
To assess the antagonistic effects, the dual culture technique was used, as previously described (Vethavalli & Sudha, 2012). A mycelial disc of 6 mm diameter was cut from the periphery of both antagonist cultures and the test pathogen and placed on a Petri plate with PDA media. For the control, only the test pathogen was placed in the centre of a Petri plate. The Petri plates were incubated at 35°C in darkness.

Data analysis
The inhibition percentage of mycelial growth= [(Gc-Gt)/Gc] × 100; Where, Gc = Mycelial growth in terms of colony diameter in control set, Gt= Mycelial growth in terms of colony diameter in treatment set. The inhibition percentages of Fusarium species growth were calculated using the following formula: Inhibition percentage (%) =100× (dc-dt)/dc; Where, dc = radial growth of pathogen in control, dt = radial growth of pathogen in dual culture. Mean values were compared through least significant different test using SAS software, version 9.4M5 (SAS Inc., Cary, NC, USA). All the experiment and test were replicated thrice.

Results
In vitro screening of extracts presenting antifungal activity All plant extracts showed a degree of growth inhibition of the tested fungus at the same concentrations. The highest inhibition of growth of the isolates was observed at 68.1% of mycelium on Coccinia grandis, which was followed by 64.1% on Allium sativum, in comparison to the control culture (100.0%). Hibiscus rosa-sinensis showed the lowest inhibition of mycelium with 29.6% against the fungal isolate in comparison to the control. The results are presented in Figure 1 and Figure 2.

Determination of minimum inhibitory concentration (MIC)
The tested plants extracts showed the MIC values ranges from 80-150µg/ml against the fungus Fusarium concentricum growth. Highest MIC value of isolated fungus was 80µg/ml on Coccinia grandis plant extract and lowest MIC value of isolated fungus was 150 µg/ml on Hibiscus rosa-sinensis plant extract (Figure 3).

In vitro antagonistic assay
The highest percentage inhibition of radial growth was observed with Trichoderma viride (46.01%) against Fusarium concentricum which was followed by 43.33% and 32.05% on Escherichia coli and Rhizobium phaseoli, respectively ( Figure 4). The antagonistic agent Rhizobium leguminosarum did not show any inhibitory activity against the isolated fungus ( Figure 4). The control group also did not show any inhibition of radial growth of Fusarium concentricum.

Discussion
Fruit rot caused by Fusarium species is very common in Bangladesh. The main objective of the present study was to study biological control measures for this fungus. Plant extracts are now a superior choice to control different plant pathogens, as reported by several previous studies (Hasan et al., 2020b;Kareem & Al-Araji, 2017;Parveen et al., 2014). In our study, we found that the plant extracts Allium sativum, Zingiber officinale, and Coccinia grandis have significant inhibitory effects on mycelial growth of Fusarium concentricum. Hosen & Shamsi (2019) also found significant antifungal activity using Allium sativum (53.85%), Ocimum sanctum (48.72%) and Zingiber officinale (49.35%) against Fusarium solani and F. oxysporum. Our current  In the current investigation, there are some limitations. Although this study showed that some plant extracts and nonpathogenic microbes could control the fungal stain, the number of plant extracts and microbes was limited. In addition, we used only methanol solvent for extraction and did not use other extractions. Moreover, we performed only in vitro techniques for antifungal potency screening and did not use any in vivo techniques. Therefore, we need to perform further studies to detect ecofriendly control this devastating fungal stain in the future.

Conclusions
We evaluated different biological control measures for the devastating Fusarium fungi. Various medicinal plants extracts and non-pathogenic microbes showed promising inhibitory activities on Fusarium concentricum in vitro. These identified control measures of Fusarium concentricum shows the importance of further research on Fusarium taxonomy to decline the risk of Fusarium concentricum caused fruit rot in Bangladesh.

Data availability
Underlying data Figshare: Effect of plant extracts on inhibition of mycelial growth of the Fusarium species. https://doi.org/10. 6084/ m9.figshare.13096262 (Hasan, 2020a). This project contains the images of Petri plates for each treatment condition.
Figshare: Effect of antagonistic agents on inhibition of mycelial growth of the Fusarium species. https://doi.org/10.6084/ m9. figshare.13096325 (Hasan, 2020b). This project contains the images of Petri plates for each treatment condition.

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